title: RNAi-mediated suppression of endogenous storage proteins leads to a change in localization of overexpressed cholera toxin B-subunit and the allergen protein RAG2 in rice seeds.
abstract: RNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2. A purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2.
authors.affiliation: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
authors.forename: Shiho
authors.initials: S
authors.lastname: Kurokawa
authors.forename: Masaharu
authors.initials: M
authors.lastname: Kuroda
authors.forename: Mio
authors.initials: M
authors.lastname: Mejima
authors.forename: Rika
authors.initials: R
authors.lastname: Nakamura
authors.forename: Yuko
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sagara
authors.forename: Natsumi
authors.initials: N
authors.lastname: Takeyama
authors.forename: Shigeru
authors.initials: S
authors.lastname: Satoh
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiyono
authors.forename: Reiko
authors.initials: R
authors.lastname: Teshima
authors.forename: Takehiro
authors.initials: T
authors.lastname: Masumura
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Yuki
mesh_terms: D000485:Allergens / Q000378:metabolism* / Q000648:ultrastructure; D002772:Cholera Toxin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D018506:Gene Expression Regulation, Plant; D005983:Glutens / Q000378:metabolism; D012275:Oryza / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D055314:Seed Storage Proteins / Q000378:metabolism*; D012639:Seeds / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000485:Allergens; D012333:RNA, Messenger; D055314:Seed Storage Proteins; D005983:Glutens; D002772:Cholera Toxin